Using molecular, biochemical and structural approaches, we have helped define how human BER proteins recognize and coordinately process target lesions. The research has centered mostly on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a non-enzymatic scaffold that facilitates the efficient execution of single-strand break repair. Some of the key mechanistic findings include: (i) in addition to abasic sites in conventional DNA, APE1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and APE1 can endonucleolytically destroy damaged RNA; (ii) APE1 contributes to the repair of 3-modifications in DNA, such as mismatches, phosphate, phosphogycolate and tyrosyl residues; (iii) the DNA repair function of APE1 is regulated in part by S-glutathionylation; (iv) inhibition of APE1 is a potential mechanism for the co-carcinogenic effects of lead, an important environmental toxin; (v) APE1 communicates with CSB, a protein defective in the premature aging disorder, Cockayne syndrome; (vi) XRCC1 directly associates with the replication/repair protein, PCNA, establishing a novel link between DNA repair and replication factories; (vii) XRCC1 coordinates disparate responses and multi-protein repair complexes that are dependent on the context of the DNA damage; (viii) the different regions of XRCC1 play distinct roles in coordinating repair complex assembly; (ix) the interaction of XRCC1 with the DNA repair enzyme PNKP functions to retain XRCC1 at DNA damage sites and promote repair of alkylation damage; (x) XRCC1 supports an emerging pathway for uracil repair, termed replication-associated BER, through a physical association with UNG2, the major nuclear uracil DNA glycosylase; (xi) the DNA glycosylase NEIL1 recognizes interstrand crosslinks in DNA, and can obstruct the efficient removal of these lethal adducts; (xii) the flap-endonuclease FEN1 plays a role in repairing mitochondrial oxidative DNA damage through a long-patch BER pathway; (xiii) RECQL4, a human RecQ helicase mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome, regulates BER capacity both directly and indirectly; (xiv) RECQL5, another RECQ helicase family member, modulates and participates in BER of endogenous DNA damage, promoting chromosome stability in normal human cells; (xv) the multifunctional protein nucleophosmin (a.k.a., NPM1) is a modulator of BER capacity by controlling protein levels and nucleolar localization of several BER enzymes; and (xvi) DNA polymerase is not only a major repair enzyme in the nucleus, but plays a role in mitochondrial maintenance in certain tissue/cell types. A main basic science focus of the lab is to establish genetically modified cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability, genome maintenance, and protection against DNA-damaging agents. Defining which of the many reported functions of APE1 are critical to normal cellular activity is a key step towards understanding the potential relationship of the protein to the aging process and disease risk. We have pioneered efforts to delineate the impact of amino acid variants found in BER proteins and to develop assays to determine the extent of inter-individual variation in BER effectiveness. Our published work has found that (i) of the three common amino acid variants of XRCC1 (i.e., R194W, R280H and R399Q), the latter two, particularly R280H, exhibit impaired recruitment to and/or retention at sites of DNA damage in live cells and (ii) variants of NEIL1 (i.e., G83D, C136R, and E181K) display altered responses to localized DNA damage in human cells. In addition, using established biochemical assays, our results indicate that for AP site incision, there exists a significant 1.9-fold inter-individual variation in repair capacity among the individuals examined. For gap-filling and nick ligation, an 1.3-fold and 3.4-fold inter-individual variation was observed, respectively. We are currently designing more sophisticated strategies to evaluate the relationship of BER capacity with disease susceptibility and premature aging phenotypes. Regarding missense variants in APE1, we have reported that except for the endometrial cancer-associated variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit largely normal structural and functional properties. The R237C mutant, conversely, displays reduced AP-DNA complex stability, 3'-5' exonuclease activity and 3'-damage processing. Our investigations have also discovered that the incision activity of R237C, in comparison to the wild-type protein, is uniquely hypersensitive to nucleosome complexes around the AP site. These findings suggest that APE1 has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes, and indicate that R237C may have contributed to carcinogenesis as an impaired-function allele. We recently reported that the tumor-associated R237C variant also shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1-deficient mouse embryonic fibroblasts. Coupled with other experimental analyses, we conclude that: (i) the tumor-associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells. Many current strategies to eradicate cancer cells employ agents that generate DNA lesions that induce cell death by blocking replication of rapidly dividing cells. Thus, a goal has been to strategically regulate the repair capacity of cancer and/or normal cells to improve the efficacy of certain therapeutic paradigms. Our results indicate that APE1, and BER more generally, is a target for inactivation in anti-cancer treatment paradigms involving alkylating drugs (e.g., temozolomide) and antimetabolites (e.g., 5-fluorouracil). Moreover, we have found that BER, and APE1 in particular, are promising targets for treating cancers with a deficiency in homologous recombination via an approach that involves synergistic lethality, and that FEN1 is a biomarker in breast and ovarian epithelial cancer. Towards the development of small molecule inhibitors against enzymes in BER, we have developed a panel of complementary and improved miniaturized high-throughput screening and profiling assays. These assays have permitted the identification of novel APE1-targeted bioactive endonuclease inhibitors, which we are working to optimize with the long-term goal of creating high affinity, selective inhibitors with therapeutic value. In addition, our effort has uncovered a set of compounds that impair the APE1/NPM1 interaction in living cells, with some of these molecules displaying anti-proliferative activity and increased cellular sensitization to therapeutically relevant genotoxins. Finally, in a separate line of investigation, we have found that serum APE1 protein levels can be used as a biomarker for predicting the prognosis and platinum-based therapeutic efficacy for non-small cell lung cancer patients.